La methionyl-ARNt synthétase (MetRS) de Saccharomyces cerevisiae aminoacyle les ARNt méthionine initiateur et élongateur (ARNtiMet et ARNteMet), mais possède également des fonctions atypiques. Nous avons montré que la MetRS rejoint le noyau durant la transition diauxique afin de réguler la transcription des gènes nucléaires des complexes III et V de la chaîne respiratoire mitochondriale. Pour ce faire, la MetRS possède au moins deux signaux de localisation nucléaire (NLS) dans sa séquence, l’un se situant dans les 55 premiers acides aminés (aa) et le second, au delà de la partie N-terminale lui permettant de recruter les sous-unités Rpb4 et Rpb7 de l’ARN pol II. Nous avons montré qu’en fermentation, la MetRS est clivée entre le 114ème et le 132ème aa et que cette forme clivée est essentielle à la viabilité des cellules, puisqu’un variant non clivé (MetRSK11A) ne permet pas la croissance. Nous avons surproduit et purifié un mutant de la MetRS clivée (MetRSΔ142) et montré que ce variant est plus efficace pour l’aminoacylation de l’ARNtiMet que la forme entière de MetRS. Ainsi, notre étude suggère que chez S. cerevisiae, la forme longue de MetRS cytoplasmique permet l’aminoacylation de l’ARNteMet, la forme longue de MetRS nucléaire régule la transcription, et la forme clivée de MetRS nucléaire et cytoplasmique permet l’aminoacylation de l’ARNtiMet / Methionyl-tRNA synthetase (MetRS) is the enzyme in charge of aminocylation of tRNA methionine initiator and elongator (tRNAiMet et tRNAeMet), but also displays atypical functions in Saccharomyces cerevisiae. In the present work, we showed that MetRS is imported to the nucleus during the diauxic shift in order to regulate transcription of genes coding for the complexes III and V subunits of the mitochondrial respiratory chain. To do so, MetRS harbors at least two nuclear localization signals (NLS), located within the 55 first aminoacids (NLS1) and beyond the N-terminal part (NLS2). The N- terminal part is responsible for the recruitment of RNA pol II subunits Rpb4 and Rpb7. We also showed that MetRS is cleaved through the 114th and the 132nd aminoacid during fermentation and that the proteolysed form is essential for the viability of the cell, since a mutant of MetRS which is not cleaved (MetRSK11A) did not allows the growth. We showed that an overproduced and purified a mutant representative of the cleaved form (MetRSΔ142) is more efficient for tRNAiMet aminoacylation than the full length MetRS. Thus, our study suggests that in S. cerevisiae, the cytoplasmic full length MetRS aminoacylates tRNAeMet, the nuclear full length MetRS regulates genes transcription, and the cytoplasmic and nuclear cleaved MetRS aminoacylates the tRNAiMet.

Les complexes multisynthétasiques (MSC) sont des complexes multi-protéiques identifiés dans un grand nombre d’organismes pro- et eucaryotes. Ils impliquent des protéines d’assemblages et des aminoacyl-ARNt synthétases (aaRSs), responsables de l’aminoacylation de leurs ARNts homologues au cours de la traduction. La taille et la composition des MSC varient selon les organismes, et le rôle de ces complexes n’est pas encore totalement compris. Il semblerait néanmoins que chez les eucaryotes, l’accrétion en complexe soit une stratégie mise en oeuvre par les cellules pour empêcher les aaRSs d’assurer des fonctions additionnelles. Chez S.cerevisiae,nous montrons que la dynamique du complexe AME, composé de la méthionyl- et de la glutamyl-ARNt synthétase (MRS et ERS) ainsi que de la protéine d’ancrage Arc1p, est dépendante du métabolisme de la levure. En respiration la MRS joue le rôle de facteur de transcription et régule l’expression des gènes nucléaires du complexe III et V de la chaîne respiratoire, tandis que l’ERS active la traduction mitochondriale. Cette étude montre que la relocalisation synchrone est primordiale pour l’adaptation des cellules au métabolisme respiratoire. / Multisynthetase complexes (MSC) are complexes made of several proteins and were identified in a wide variety of organisms from pro- to eukaryotes. They are usually made of assembly factors and aminoacyl-tRNA synthetases (aaRSs), which are responsible for the aminoacylation of their corresponding tRNAs during translation. Depending on the organisms, size and composition of these complexes differ greatly and their role is not fully understood yet. Although it seems that in eukaryotes, accretions of aaRSs into MSC prevent aaRSs to perform their additional functions. In the yeast Saccharomyces cerevisiae, we show that the dynamic of the AME complex, made of the méthionyl- and glutamyl-tRNA synthetases (MRS and ERS) and the assembly protein Arc1p is linkedto yeast metabolism. In respiration, MRS is imported in the nucleus to act as a transcription factor and regulates the expression of nuclear genes belonging to complex III and V of the respiratory chain, while ERS is imported in mitochondria to activate translation. This study shows that synchronous relocation of both aaRSs is crucial for yeast cells to adapt to respiratory metabolism.

CSIR / Translation initiation is a rate limiting step during protein biosynthesis. Initiation occurs by formation of an initiation complex comprising 30S subunit of ribosome, mRNA, initiator tRNA, and initiation factors. The initiator tRNA has a specialized function of binding to ribosomal P site whereas all the other tRNAs are selected in the ribosomal A site. The presence of a highly conserved 3 consecutive G-C base pairs in the anticodon stem of the initiator tRNA has been shown to be responsible for its P-site targeting. The exact molecular mechanism involved in the P-site targeting of the initiator tRNA is still unclear and focus of our study. Using genetic methods, we obtained mutant E. coli strains where initiator tRNA mutants lacking the characteristic 3-GC base pairs can also initiate translation. One such mutant strain, A30, was selected for this study. Using standard molecular genetic tools, the mutation was mapped and identified to be a mutation in a transcription remodeling factor, RapA (A511V). RapA is a transcription recycling factor and it displaces S1 when it performs its transcription recycling activity. We found this mutation to cause an increase in the S1-depleted ribosomes leading to decreased fidelity of translation initiation as the mutant RapA inefficiently displaces S1 from RNA polymerase complex. The mutation in the RapA was also found to cause changes in the transcriptome which leads to downregulation of major genes important for methionine and purine metabolism. Using mass spectrometric analysis, we identified deficiencies of methionine and adenine in the strain carrying mutant RapA. Our lab had previously reported that methionine and S-adenosyl methionine deficiency cause deficiency of methylations in ribosome which in turn decreases the fidelity of protein synthesis initiation. We used strains deleted for two newly identified methyltransferases, namely RlmH and RlmI, for our study and these strains also showed decreased fidelity of initiation. RlmH and RlmI methylate 1915 and 1962 positions of 23S rRNA respectively. We found that deletion of these methyltransferases also caused defects in ribosome biogenesis and compromised activity of ribosome recycling factor. We constructed phylogenetic trees of the initiator tRNA from 158 species which distinctly assembled into three domains of life. We also constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNAPro, tRNAGlu, or tRNAThr (but surprisingly not elongator tRNAMet) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the three domains of life. Our results indicate that methionine selection, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. In conclusion, the current study reveals the importance of methylations in ribosome biogenesis and fidelity of translation initiation. It also strongly suggests a co-evolution of the metabolism and translation apparatus giving adaptive advantage to the cells where presence of methionine in the environment can be a signal to initiate translation with methionine initiator tRNA.

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Background: To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology. Results: Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced. Conclusions: These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.

Background: Transfer RNAs (tRNAs) are ubiquitous in all living organism. They implement the genetic code so that most genomes contain distinct tRNAs for almost all 61 codons. They behave similar to mobile elements and proliferate in genomes spawning both local and non-local copies. Most tRNA families are therefore typically present as multicopy genes. The members of the individual tRNA families evolve under concerted or rapid birth-death evolution, so that paralogous copies maintain almost identical sequences over long evolutionary time-scales. To a good approximation these are functionally equivalent. Individual tRNA copies thus are evolutionary unstable and easily turn into pseudogenes and disappear. This leads to a rapid turnover of tRNAs and often large differences in the tRNA complements of closely related species. Since tRNA paralogs are not distinguished by sequence, common methods cannot not be used to establish orthology between tRNA genes. Results: In this contribution we introduce a general framework to distinguish orthologs and paralogs in gene families that are subject to concerted evolution. It is based on the use of uniquely aligned adjacent sequence elements as anchors to establish syntenic conservation of sequence intervals. In practice, anchors and intervals can be extracted from genome-wide multiple sequence alignments. Syntenic clusters of concertedly evolving genes of different families can then be subdivided by list alignments, leading to usually small clusters of candidate co-orthologs. On the basis of recent advances in phylogenetic combinatorics, these candidate clusters can be further processed by cograph editing to recover their duplication histories. We developed a workflow that can be conceptualized as stepwise refinement of a graph of homologous genes. We apply this analysis strategy with different types of synteny anchors to investigate the evolution of tRNAs in primates and fruit flies. We identified a large number of tRNA remolding events concentrated at the tips of the phylogeny. With one notable exception all phylogenetically old tRNA remoldings do not change the isoacceptor class. Conclusions: Gene families evolving under concerted evolution are not amenable to classical phylogenetic analyses since paralogs maintain identical, species-specific sequences, precluding the estimation of correct gene trees from sequence differences. This leaves conservation of syntenic arrangements with respect to "anchor elements" that are not subject to concerted evolution as the only viable source of phylogenetic information. We have demonstrated here that a purely synteny-based analysis of tRNA gene histories is indeed feasible. Although the choice of synteny anchors influences the resolution in particular when tight gene clusters are present, and the quality of sequence alignments, genome assemblies, and genome rearrangements limits the scope of the analysis, largely coherent results can be obtained for tRNAs. In particular, we conclude that a large fraction of the tRNAs are recent copies. This proliferation is compensated by rapid pseudogenization as exemplified by many very recent alloacceptor remoldings.

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The ribosome is one of the most universal molecular machinery, synthesizing proteins in all living systems. The small ribosomal subunit plays a crucial role in decoding the messenger RNA during translation. We propose and validate a new architectural model of the ribosomal small subunit, with broad implications for function, biogenesis and evolution. We define an rRNA domain: compact and modular, stabilized by self-consistent molecular interactions, with ability to fold autonomously when it is isolated from surrounding RNA or protein. Each rRNA helix must be allocated uniquely to a single domain. These criteria identify a core domain of small subunit rRNA (domain A), which acts as a hub, linking to all other domains by A-form helical spokes. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2’OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with autonomous folding, and therefore classification as a domain. We show that the domain concept is applicable and useful for understanding the small ribosomal subunit. Our results support the utility of the concept of the domain as applied to at least some RNAs, the interdependence of the elements of domain A, and its ability to fold autonomously. Moreover, domain A, which exhibits elements of tRNA mimicry, is the essential core of the small ribosomal subunit. Understanding the structure and dynamics of domain A will provide valuable insight into the translational machinery.